A role for intracellular zinc in glioma alteration of neuronal chloride equilibrium

Glioma patients commonly suffer from epileptic seizures. However, the mechanisms of glioma-associated epilepsy are far to be completely understood. Using glioma-neurons co-cultures, we found that tumor cells are able to deeply influence neuronal chloride homeostasis, by depolarizing the reversal potential of γ-aminobutyric acid (GABA)-evoked currents (EGABA). EGABA depolarizing shift is due to zinc-dependent reduction of neuronal KCC2 activity and requires glutamate release from glioma cells. Consistently, intracellular zinc loading rapidly depolarizes EGABA in mouse hippocampal neurons, through the Src/Trk pathway and this effect is promptly reverted upon zinc chelation. This study provides a possible molecular mechanism linking glioma invasion to excitation/inhibition imbalance and epileptic seizures, through the zinc–mediated disruption of neuronal chloride homeostasis.

Glioma-associated epilepsy is an established but poorly understood phenomenon. Over 80% of glioma patients suffer from seizures, 1 often representing the first symptomatic presentation of a tumor and possibly preceding it. 2 It has been extensively reported that glioma cells release glutamate in the extracellular space, through the glutamate-cystine transporter (system Xc), promoting proliferation and invasion and causing neuronal death. 3 Accordingly, increased glutamate levels have been implicated in numerous seizure disorders 4 and contribute to epileptogenesis in gliomaimplanted rodents. 5,6 Glutamate excess may cause the alteration of neuronal chloride (Cl − ) homeostasis and depolarize γ-aminobutyric acid (GABA) reversal potential (E GABA ), as a result of Cl − transporters dysfunction or disequilibrium. 7 Indeed, a precise balance between NKCC1 and KCC2 activity is necessary for inhibitory GABAergic signaling in the adult central nervous system 8 and its disequilibrium can cause elevation of intracellular [Cl − ] leading to switch of GABAergic signaling from hyperpolarizing to depolarizing in epileptic tissue, 9,10 contributing to epileptogenesis. [11][12][13] In this study, we investigated the mechanisms of gliomainduced neuronal overexcitation using co-cultures of hippocampal and glioma cells. We report that glioma cells cause the alteration of E GABA , through glutamate-receptor-dependent zinc (Zn 2+ ) accumulation, leading to KCC2-mediated Cl − transport unbalance. Our study provides the molecular mechanism of glioma-induced elevation in intracellular Cl − and a complete model linking glutamate release by glioma cells to glioma-related epilepsy.

Results
Glioma co-culture increases neuronal [Cl -] i by a glutamatergic mechanism. To address the effect of glioma cells on neuronal Cl − equilibrium, we determined the reversal potential of the currents evoked by GABA application in mouse hippocampal cultured neurons. As shown in Figures  1a and b, co-culturing neurons with patient-derived glioma cells (MZC) 14 caused a rightward shift in the current-voltage relationship of GABA-mediated responses, giving a positive shift of E GABA from − 73.9 ± 1.2 mV (control; n = 124) to − 52.1 ± 1.6 mV (co-culture; n = 101, Po0.001).
In control neurons, E GABA was significantly below resting membrane potential (RMP); conversely, in co-cultured neurons, despite a small depolarization of resting potential (Figure 1b), E GABA value was consistently more positive than RMP, reverting the driving force for GABA-mediated currents ( Figure 1c). Similar results were observed also after a shorter (4h) co-culture duration (E GABA = − 52.2 ± 4.4 mV; n = 18). E GABA shift resulted from the increase in [Cl − ] i , as directly demonstrated using a genetically encoded Cl-Sensor, 15,16 which gave values similar to those calculated by Nernst equation (Figure 1d).
In addition, when the system Xc blocker sulfasalazine (250 μM) was added to the co-culture medium, E GABA depolarization was prevented (Figure 2a), showing that this effect requires Xc-mediated glutamate release.
Moreover, acutely applied glioma-conditioned medium (GCM) activated APV/NBQX-sensitive inward currents ( Figure 2b) in hippocampal neurons, confirming the presence of GluRs agonists in GCM. 17 Altogether, these data show that in the co-culture conditions, glioma cells cause the depolarizing shift of Cl − equilibrium potential in neurons, through glutamate release and GluRs activation. Consistently, E GABA depolarization was observed in neurons co-cultured with different human and murine glioma cells, but not with astrocytes (Figure 2c, and  Supplementary Table S1), indicating a tumor-specific effect.
The alteration of Cl − transporter activity is involved in glioma-induced E GABA shift in co-culture. In fact, in co-cultured neurons, furosemide, bumetanide or DIOA treatment abolished E GABA depolarization ( Figure 3b). However, the acute application of bumetanide reverted the glioma-induced effect   Figure 3b). These data indicate that glioma-induced E GABA depolarization is due to the unbalance of cation-chloride transporters activity, likely due to KCC2 reduced function.
To disclose the effects of glioma co-culture on neuronal KCC2 protein expression, we performed immunoblot experiments, revealing that the level of transporter expression was similar in control and co-cultured neurons (Figure 3d), thus indicating that glioma-induced alteration of neuronal Cl − homeostasis relies on functional KCC2 modulation, rather than on changes in expression.
Using FluoZin-based fluorescence determinations, we also observed that basal intracellular Zn 2+ was significantly higher in co-cultured neurons in respect to control (Figure 4b). Neuronal Zn 2+ accumulation was prevented by APV/NBQX application in the co-culture medium (Figure 4c), indicating the requirement for GluR activation. Consistently, the application of GCM or glutamate (20 μM) onto FluoZin-loaded neurons, elicited an APV/NBQX-sensitive fluorescence increase, rapidly reverting to basal level upon TPEN application (Supplementary Figure S1 and S2).
To identify the source of Zn 2+ , we performed experiments in the presence of tricine, a chelator of extracellular Zn 2+ . Tricine treatment (1 mM, 24 h) did not abolish the effect of co-culture on E GABA (Supplementary Table S2), indicating that extracellular Zn 2+ is not required for E GABA depolarization. However, co-culture induced [Zn 2+ ] i accumulation was not observed (not shown). Noteworthy, tricine treatment caused a reduction in basal Zn 2+ both in control and co-cultured neurons (n = 41/59, control/co-culture; not shown). These results suggest that extracellular Zn 2+ chelation modifies neuronal Zn 2+ homeostasis, altering basal cytosolic Zn 2+ level. To avoid possible effects on intracellular Zn 2+ homeostasis, experiments with Zn 2+ chelators were repeated reducing incubation time (4 h), following the observation that 4 h tricine treatment did not impair neuronal ability to release intracellular Zn 2+ in response to a glutamatergic stimulus (Supplementary Figure  S2). Consistently, tricine did not prevent the depolarizing effect of 4 h co-culture on E GABA (Figure 4d). Conversely, when intracellular Zn 2+ was chelated (with FluoZin 5 μM, 4 h), coculture-induced E GABA shift was abolished ( Figure 4d).
All together, these data indicate that Cl − disequilibrium in co-cultured neurons is due to intracellular Zn 2+ -dependent KCC2 impairment.
Zn 2+ -mediated E GABA shift requires Src/TrkB activation. To disclose the mechanisms underlying Zn 2+ -mediated E GABA shift, neuronal [Zn 2+ ] i was artificially increased through perforated (by gramicidin) patch pipette loading. We preliminarily verified the efficacy of intracellular Zn 2+ loading through perforated patch by fluorescence recordings; the presence of ZnCl 2 in the pipette solution caused a time-and concentration-dependent fluorescence increase in FluoZinloaded neurons, indicating that, in our experimental conditions, Zn 2+ permeates through gramicidin pores (Supplementary Table S3 Figure 5b) rapidly reverted Zn 2+ -induced E GABA depolarization on control neurons. These data indicate that intracellular Zn 2+ level rapidly and reversibly interferes with Cl − equilibrium, through KCC2 activity modulation, highlighting intracellular Zn 2+ rise as the key step in gliomainduced E GABA depolarization. Several mechanisms have been proposed to explain KCC2 downregulation in hyperexcitability models, 7,20,21 including the phosphorylation of KCC2 residues by a number of different kinases. To explore the mechanism of Zn 2+ -mediated E GABA shift, we investigated the involvement of Src/TrkB-dependent KCC2 tyrosine phosphorylation, 20,21 as intracellular Zn 2+ has been reported to transactivate TrkB in a Src-dependent manner. 22 When hippocampal cultures were treated with TrkB inhibitor K252A (200 nM; 1 h pre-application and perfused  Table S4). Similarly, in the presence of Src kinase inhibitor PP2 (5 μM; 1 h pre-application and perfused during the experiment), E GABA shift due to intracellular Zn 2+ loading was absent (Figure 5c  and Supplementary Table S4). Thus, Zn 2+ -induced E GABA shift requires the integrity of Src/TrkB pathway. Consistently, by western blots analysis, we demonstrated that GCM treatment (15 min) significantly increased neuronal Src phosphorylation (Figure 5d). This effect was Zn 2+ dependent, as it was prevented by TPEN application (20 μM, 15 min pre-treatment and during GCM application, n = 6; P = 0.92 with respect to TPEN, Figure 5e). Moreover, GCM treatment significantly increased neuronal TrkB phosphorylation ( Figure 5f).
Altogether, these data indicate that glioma-released factors might alter neuronal Cl − homeostasis through Zn 2+ -induced Src/TrkB-mediated KCC2 modulation, as illustrated in Figure 6.

Discussion
We used co-cultures of hippocampal neurons and glioma cells to unveil the mechanisms of glioma-induced hyperexcitability, reporting that glioma cells depolarize neuronal E GABA , increasing [Cl − ] i and reverting the driving force for GABA-mediated currents. Our results show that E GABA depolarization relies on Zn 2+ -mediated KCC2 functional impairment, disclosing the underlying mechanism: glioma-released glutamate activates neuronal GluRs, causing neuronal intracellular Zn 2+ rise which, through Src/TrkB activation, reduces KCC2 activity, leading to intracellular [Cl − ] increase and E GABA depolarization. We conclude that glioma might reduce neuronal inhibition through Zn 2+ -mediated downregulation of KCC2 activity, causing hyperexcitability.
In glioma-co-cultured hippocampal neurons, the currentvoltage relationship of GABA-mediated responses is shifted to more depolarized potentials, compared with control, giving a more depolarized E GABA . This indicates a higher basal neuronal [Cl − ] i in co-cultures, confirmed by an independent estimation in neurons transfected with a YFP-based Cl − Sensor. Although glioma co-culture induces a small neuronal depolarization, the shift of E GABA is more relevant, resulting in the inversion of the driving force for GABA-mediated currents.
According to previous studies, Cl − homeostasis in cultured hippocampal neurons is determined by the activity of both NKCC1 and KCC2. 8 Indeed, we show here that both transporters are expressed in control neurons and their activity is required to maintain Cl − equilibrium as blocking either NKCC1 or KCC2 leads to a shift in basal E GABA . Conversely, in co-cultured neurons, NKCC1 activity is (ii) the specific NKCC1 antagonist bumetanide reverts coculture-induced E GABA depolarization, demonstrating that Cl − accumulation requires the activity of NKCC1; (iii) neurons treated with the specific KCC2 blocker DIOA display depolarized E GABA , likely occluding the co-culture effect. Altogether, these data support the proposal that in neurons, a new Cl − equilibrium is established during glioma co-culture, characterized by higher [Cl − ] i and caused by reduced KCC2 activity. We provide evidence that glioma-induced KCC2 impairment, observed in co-cultured neurons, is due to intracellular Zn 2+ rise. 18 Indeed, intracellular Zn 2+ chelation by TPEN rapidly reverts co-culture-induced E GABA shift and basal [Zn 2+ ] i is significantly higher in neurons after glioma co-culture.
Glioma-induced alteration of neuronal Cl − homeostasis likely depends on functional KCC2 block, rather than on reduction of protein expression (see Lee et al. 7 ). This view is based on the unaltered expression of neuronal KCC2 after glioma co-culture and on the rapid rescue exerted by Zn 2+ chelation, demonstrating a dynamic modulation of Cl − transport mechanisms. The functional modulation of KCC2 activity has been observed both in physiological and pathological models, such as prolonged post-synaptic spiking, 25 brainderived neurotrophic factor (BDNF) stimulation 26 and oxygen glucose deprivation. 18 Our data suggest that the mechanism responsible for glioma-induced KCC2 inhibition relies on Zn 2+mediated Src/TrkB activation. Indeed, Zn 2+ -induced E GABA shift was prevented by the application of Src or TrkB kinase inhibitors (PP2 or K252A). Consistently, GCM increased the level of TrkB and Src phosphorylation in hippocampal cultures, the latter effect being prevented by the Zn 2+ chelator TPEN. These results are in line with the notion that intracellular Zn 2+ may transactivate TrkB by a neurotrophin-independent and Src-dependent mechanism, as reported in models of intense neuronal activity. 22 In neurons, Zn 2+ -induced TrkB transactivation may mimic BDNF-TrkB signaling, leading to KCC2 phosphorylation on tyrosine residues 27,28 and driving neuronal disinhibition. 29 We can speculate that co-culture induced E GABA depolarization needs KCC2 phosphorylation on tyrosine residues because of Zn 2+ -induced TrkB transactivation.
We report that glioma-induced E GABA shift requires the release of glutamate in the extracellular space by glioma cells and the consequent activation of neuronal ionotropic GluRs. Indeed, the application of APV and NBQX during co-culture prevents E GABA depolarization.
It is known that glutamate can induce intracellular Zn 2+ increase through different mechanisms, including AMPARs and Ca 2+ channel-mediated influx or Ca 2+ -dependent intracellular release. 25,30 We observed that both APV and NBQX abolished co-culture-induced E GABA depolarization highlighting the role for both ionotropic GluRs in this effect. The simplest explanation for this evidence is that AMPARmediated neuronal depolarization drives NMDAR activation thus allowing Ca 2+ -dependent intracellular Zn 2+ release. 30 Our data indicate that the source of Zn 2+ is intracellular, as the extracellular Zn 2+ chelator tricine was ineffective, whereas FluoZin prevented co-culture-induced E GABA depolarization.
It has to be considered that tricine treatment, although did not prevent co-culture-induced E GABA depolarization, inhibited co-culture-induced basal [Zn 2+ ] i accumulation. Thus, perturbing extracellular Zn 2+ concentration may modify neuronal Zn 2+ homeostasis, preventing cytosolic Zn 2+ accumulation. However, tricine-treated neurons retained the ability to release intracellular Zn 2+ in response to a glutamatergic stimulus, and this event is likely sufficient to trigger the intracellular signaling leading to KCC2 impairment.
It is well established that intracellular Zn 2+ rise induces cell death, and Zn 2+ exposure is toxic to neurons both in vitro and in vivo. It is now evident that increased cytosolic Zn 2+ resulting from liberation from intracellular stores, rather than cytoplasmic influx of synaptically released Zn 2+ , can be highly toxic during oxidative and other types of neuronal injury, 31 and Zn 2+ dyshomeostasis appears to be a common feature of numerous neuropathological conditions. 23,32 We speculate that the reported mechanism, leading to reduced GABAergic transmission, could underlie the etiology of glioma-related epilepsy, pointing to Zn 2+ accumulation as a possible therapeutic target to restore KCC2 function and the excitatory/inhibitory balance. In this view, it is possible to speculate that Zn 2+ homeostatic drugs may be helpful in the treatment of Zn 2+ -related neurological disorders such as neuronal hyperexcitability or Alzheimer's disease. 33 The use of co-cultures 34 allowed to disclose the molecular mechanisms involved in glutamate-mediated overexcitation induced by glioma. Our results are in line with recent works showing that Xc-mediated glutamate release is responsible for the generation of tumor-associated epileptic events in gliomabearing mice. 6,35 Indeed, increased concentration of extracellular glutamate has been found in peritumoral tissue in both humans and mice, 6,36 supporting its role in tumor growth, survival and peritumoral seizure activity. 6 Additional sources of glutamate in peritumoral tissue may be microglial Xc system or reverse activity of Na + -dependent glutamate transporters in neurons or astrocytes. 37,38 Thus, the co-culture system likely retains the feature of excessive glutamate release typical of glioma. Consistently, neuronal E GABA shift was observed in coculturing neurons with different glioblastoma cell lines, but not with astrocytes, supporting the view of a tumor-specific effect. 6 Glutamate-induced alteration of neuronal Cl − homeostasis may act concomitantly with other mechanisms, including the direct depolarizing effect of glutamate on neurons, displacing the excitation/inhibition balance toward an increased network excitability, thus promoting seizure onset. 35 In conclusion, our study provides a possible explanation of the mechanisms by which glioma cells affect neuronal Cl − equilibrium, highlighting the role of Zn 2+ , recently emerged in a variety of excitotoxic conditions, such as epilepsy, ischemia and brain trauma. 32 Primary hippocampal neuronal cultures. Hippocampal neuronal cultures were prepared from newborn (P0-P1) C57BL/6 mice of either sex (Charles River -Research Models and Services, Lecco, Italy). In brief, after careful dissection from diencephalic structures, the meninges were removed and the hippocampi were chopped and digested in 1.25 mg/ml trypsin for 20 min at 37°C. Cells were mechanically dissociated and plated at a density of 10 5 in poly-L-lysine-coated glass coverslip (12 mm diameter) in serum-free Neurobasal Medium (Gibco Life Science, Life Technologies Italia, Monza, Italy), supplemented with B27 2 mM L-glutamine and 100 μg/ml gentamicin (neuronal culture medium). Then, cells were kept at 37°C in 5% CO 2 for 10-13 days with medium replacement (1 : 1 ratio) three times per week. With this method, we obtained cultures composed by 60-70% neurons, 30-35% astrocytes and 4-5% microglia, as determined with β-tubulin III, glial fibrillary acidic protein and isolectin IB4 staining. 38 The same procedure was followed to prepare rat hippocampal culture used for some immunoblot experiments.
Glial primary cultures. Primary cortical glial cells were prepared from P0-P2 mice. Cerebral cortices were chopped and digested in 30 U/ml papain for 40 min at 37°C and gently triturated. The dissociated cells were washed, suspended in Dulbecco's Modified Eagle Medium (Gibco, Life Technologies Italia) with Glutamax with 10% fetal bovine serum (Invitrogen, Life Technologies Italia) and plated at a density of 9-10 × 10 5 in 175 cm 2 cell culture flasks. At confluence (10-14 days in vitro, DIV), glial cells were shaken for 2 h at 37°C to detach and remove microglial cells. These procedures gave almost pure astrocytes cell population (4-6% of microglia contamination), as verified by staining with glial fibrillary acidic protein and isolectin IB4. 34 Neuronal culture transfection. Hippocampal neuronal cultures were transfected at 9-10 DIV. One day before transfection, 50% of the culture medium was replaced with fresh medium. For transfection, 100 μl of Neurobasal media were mixed with 2 μl of NeuroMag (OZ Bioscience, Marseille, France) and 1 μg of Cl-Sensor cDNA. 15,16 The mixture was incubated for 15-20 min at room temperature and thereafter distributed dropwise over the neuronal culture. Neuronal cultures were placed on a magnetic board (OZ Bioscience) and incubated for 15 min (37°C, 5% CO 2 ). One hour later, 50% of neuronal culture medium was substituted with fresh neuronal culture medium. Cells were used for experiments 24-76 h after transfection.
Co-cultures. Glioma cells or astrocytes, prepared as above, were re-suspended in neuronal culture medium and replated by seeding 10 5 cells onto 0.33 cm 2 transwell cell-culture inserts (pore size 0.4 μM, Corning B.V. Life Sciences, Amsterdam, The Netherlands), allowing traffic of small diffusible substances, but preventing cell-to-cell contact. Transwell inserts were transferred into 24-well cultures plates, containing 10-12 DIV primary hippocampal cultures in neuronal culture medium.
Neuronal viability after 4 and 24 h of co-culture was evaluated as reported in Supplementary Figure S4.
Patch-clamp recordings. Patch-clamp recordings were obtained using glass electrodes (3)(4)(5) filled with the following intracellular solution (in mM): 140 KCl, 2 MgCl 2 , 10 HEPES, 2 MgATP, 0.5 EGTA; pH 7.3, with KOH. Perforated patch-clamp recordings with access resistances between 30 and 40 MΩ were obtained using gramicidin D. 42 Gramicidin, prepared every 2 h, was added to the pipette solution to a final concentration of 50 μg/ml. During experiments, neurons were continuously superfused with normal extracellular solution containing (in mM): 140 NaCl, 2.5 KCl, 2 CaCl 2 , 2 MgCl 2 , 10 HEPES-NaOH and 10 glucose (pH 7.3), added with tetrodotoxin (0.2 μM), using a gravity-driven perfusion system, consisting of independent tubes for standard and agonist-containing solutions, connected to a fast exchanger system (RSC-100; Bio-Logic, Claix, France). All recordings were performed at 24-25°C. For I-V experiments, neurons were voltage-clamped at − 70 mV with 6-s steps to each test potential; GABA (100 μM; 500 ms; every 30 s) was applied 1 s after the voltage step onset. A linear regression was used to calculate the voltage dependence of GABA-evoked current. E GABA was taken as the intercept of this best-fit line for each cell. We minimized bicarbonate flux through the GABA A channels, using an HEPES-buffered extracellular solution (nominally CO 2and bicarbonate-free 43,44 ), so that E GABA was an estimation of E Cl . Membrane currents, recorded with a patch-clamp amplifier (Axopatch 200B; Molecular Devices, Foster city, CA, USA), were filtered at 2 kHz, digitized (10 kHz) and acquired with Clampex 10 software (Molecular Devices). The stability of the patch was checked by repetitively monitoring the input and series resistance during the experiment, and recordings were discarded when any of these parameters changed by 410%.  Supplementary Table S3.
Cell stimulation and western blot analysis. For western blot experiments, 4 × 10 5 hippocampal cells were plated on poly-L-lysine coated 12-well cultures plates. 45 For the analysis of TrkB and Src activation, cultured hippocampal neurons were incubated for 2 h in Locke's buffer and stimulated for 15 min with either GCM or drugs: BDNF (100 ng/ml), platelet-derived growth factor (100 ng/ml). To assess the role of Zn 2+ rise in Src phosphorylation, hippocampal cultures were treated with the Zn 2+ chelator TPEN (20 μM, 15 min pre-application, and during GCM stimulation). Corresponding cell lysates were run on SDS-polyacrylamide gel and analyzed for Src or TrkB phosphorylation. Densitometric analysis was performed with QuantityOne software (Bio-Rad Laboratories S.r.l., Segrate (MI), Italy) and phosphoprotein levels were normalized for TrkB and Src expression. For KCC2 expression determination, western blot analysis was performed on rat hippocampal cell lysates in control conditions and after 24 h co-culture with equal number of glioma cells. For each condition, equal amounts of proteins were loaded on SDS-PAGE gel for immunoblot analysis with rabbit anti-KCC2. Protein levels were normalized for TuJ1 expression. Drug Application. During electrophysiological and fluorescence measurements, neurons were continuously superfused with normal extracellular solution, using a gravity-driven perfusion system, consisting of independent tubes for standard and agonist-containing (GABA, GCM) solutions, connected to a fast exchanger system (RSC-100; Bio-Logic) positioned 50-100 μm from the cell. Antagonists were usually acutely applied through a parallel tubes of the same perfusion system. DIOA, K252A and PP2 were pre-incubated for 1 h (37°C) and then continuously applied during the experiments. Sulfasalazine, APV/NBQX, BAPTA-AM and tetrodotoxin citrate were applied during co-culture in the co-culture medium and not superfused during recordings (24 h). The values of EGABA and RMP of control neurons, following different drug treatments are reported in Supplementary Table S2 and Supplementary Table S4.
Data analysis. Data, analyzed offline, are presented as mean ± S.E.M.; we used the QuantityOne (Bio-Rad Laboratories S.r.l.) program for the densitometric analysis of all immunoblots. Origin 7 (Origin software; Microcal Software, Northampton, MA, USA) and Sigmaplot 11 (Systat Software Inc, London, UK) software were used for statistical analysis. Paired and unpaired t-test and one-way ANOVA were used for parametrical data, as indicated; Tukey test was used as post hoc test; Mann and Withney test for non parametrical data. We constructed I-V plots, cumulative distribution plots and fitted data points by linear or non-linear regression analysis using Origin software. Statistical significance for cumulative distributions was assessed with Kolmogorov-Smirnov test.

Conflict of Interest
The authors declare no conflict of interests.